Binding to the naturally occurring double p53 binding site of the Mdm2 promoter alleviates the requirement for p53 C-terminal activation

Abstract

Genotoxic stress activation of the tumor suppressor transcription factor p53 involves post-translational C-terminal modifications that increase both protein stability and DNA binding activity. We compared the requirement for p53 protein activation of p53 target sequences in two major p53-regulated genes, p21/WAF1 (encoding a cell cycle inhibitory protein) and Mdm2 (encoding a ubiquitin ligase that targets p53 for proteolytic degradation). The p53 binding site in the proximal p21/WAF1 promoter contains a single p53 binding consensus sequence, while the p53 binding site in the Mdm2 promoter contains two consensus sequences linked by a 17 bp spacer. Binding of recombinant p53 protein to the p21/WAF1 binding site required monoclonal antibody PAb421, which can mimic activating phosphorylation and/or acetylation events at the C-terminus. In contrast, recombinant p53 bound strongly to the Mdm2 binding site in the absence of PAb421 antibody. Separate binding to each consensus sequence of the Mdm2 binding site still required PAb421, indicating that p53 binding was not simply due to greater affinity to the Mdm2 consensus sequences. Linking two p21/WAF1 binding sites with the 17 bp spacer region from the Mdm2 gene eliminated the PAb421 requirement for p53 binding to the p21/WAF1 site. These results suggest a mechanism for regulation of Mdm2 gene transcription that differs from that other p53-induced genes by its lack of a requirement for C-terminal activation of p53 protein. A steady induction of Mdm2 protein would maintain p53 protein at low levels until post-translational modifications following DNA damage increased p53 activity towards other genes, mediating p53 growth inhibitory and apoptotic activities.

Defective p53 Post-translational Modification Required for Wild Type p53 Inactivation in Malignant Epithelial Cells with mdm2 Gene Amplification

“…2A, lanes 1-3 and 10 -12, respectively), as expected (17). Next the transcriptional activity of p53 in 03C and 03R cells was analyzed by immunoblotting for p21 protein accumulation in response to IR, and by activation of a p53-directed GFP reporter plasmid (15) in response to UV, because of the p53-independent accumulation of p21 protein in NK-1 cells in response to UV (Fig. 2A, lanes 10 -12), as has been previously described (22).…”

“…Plasmids and cDNA Constructs-The green fluorescent protein (GFP) 1 reporter plasmids were constructed by inserting either two p21-derived p53 responsive elements, pGFP-p53x2 (GCTCAAGCTTCGAA-TTCTAGAGAACATGTCCCAACATGTTGGGCGTCGGCTGTCGGGG-AACATGTCCCAACATGTTGCGGGCATTGATCCGAGGTCCACTTC-GCTATATATTCCCCGAGCTCCTATCTACACGG) (15), or mutated p53-binding sequences, pGFP-p53mut (containing MG 15 (16)), into the pEGFP-1 vector (BD Biosciences). The pCHDM1A plasmid, expressing N-terminal HA-tagged human Mdm2, was kindly provided by Hua Lu (OHSU, Portland, OR).…”

“…The overexpression of Mdm2 alone was not responsible for the continued DNA-bound p53-Mdm2 complex, because Mdm2 overexpression in 03C precancer cells did not result in Mdm2 association with DNA-bound p53. The continued association between DNA-bound p53 and Mdm2 was not the result of failed p53 phosphorylation at Ser 18 , -23, or -37 (Ser 15 , -20, and -33 in humans, respectively), which all were detected in 03R cancer cells after UV treatment. However, the exogenous expression of p53 protein mimicking phosphorylation at Thr 21 (18 in humans) prevented association of Mdm2 with DNA-bound p53, formed a DNA-bound p53-p300 complex, and induced p21 induction in response to DNA damage.…”

HLA-B-associated transcript 3 (Bat3)/Scythe is essential for p300-mediated acetylation of p53

“…Firstly, ⌬Cp53 is defective in its induction of p21 and apoptosis (Chen et al 1996). Secondly, CT modification is necessary for efficient p53 binding to the p21 promoter but not for binding to the Mdm2 promoter, presumably because these two genes have structurally different p53REs (Kaku et al 2001). Thirdly, ⌬Cp53 is much less efficient in binding to the p21 promoter than to the Hdm2 promoter, in a way that closely correlates with decreased induction of the target gene (McKinney et al 2004).…”

c-Abl-independent p73 stabilization during gemcitabine- or 4′-thio-β-d-arabinofuranosylcytosine–induced apoptosis in wild-type and p53-null colorectal cancer cells

“…3A). The Mdm2 gene contains a p53 binding site composed of two consensus sequences linked by a 17-bp spacer (44). Mdm2 is transcriptionally activated by p53 and is part of an autoregulatory feedback loop that mediates p53 ubiquitination and degradation through the proteasome pathway.…”